Isolation of Amidase-negative Mutants of Pseudomonas aeruginosa Using Glycollamide as a Selective Agent

Summary: A positive selection is described for isolating amidase-negative mutants from Pseudomonas aeruginosa strains. The method is based on the conversion, via amidase activity, of glycollamide to glycollate which is growth inhibitory. Three types of mutant were isolated on lactate medium containing glycollamide: (i) mutants in which amidase activity was reduced or absent; (ii) double mutants that were amidase-negative and resistant to glycollate inhibition of growth; and (iii) glycollate-resistant mutants. By raising glycollamide concentrations in the selection medium, amidase-negative mutants were obtained from strains producing altered amidases with low specific acetamidase and glycollamidase activities. Glycollamide has wider applicability than fluoroacetamide as a selective agent for obtaining amidase-negative mutants.